PROJECT SUMMARY/ABSTRACT There are 12-15 million fractures in the U.S. each year, which lead to 60 million work days lost, more than twice the number for heart disease and stroke combined. As many as 5-10% of fractures have delayed or failed healing, i.e., ?nonunion?. In cases of atrophic nonunion, there is a ?failure of biology? to form the normal osteochondral callus tissue that bridges the fracture gap and is essential for healing. Clinical treatment of atrophic nonunions is challenging and outcomes are unpredictable. Thus, there is an unmet clinical need for improved treatments. However, the successful development of new treatments for atrophic nonunion requires: a) a better understanding of their underlying biology, and b) the use of biologically-relevant animal models. The overall goal of this proposal is to develop new murine models of atrophic nonunion to address these issues. After fracture, periosteal progenitor cells (PPCs) rapidly proliferate, providing a source of chondrocytes and osteoblasts to the fracture callus. Surprisingly, the requirement of PPC proliferation for fracture healing has not been directly tested, and the lineage of the cells that proliferate to form fracture callus is poorly understood. We propose to target the process of PPC proliferation using a genetic approach. We will use mice carrying the herpes simplex virus-thymidine kinase (HSV-TK) ?suicide gene? to disrupt cell replication with lineage and temporal control. By itself, the TK enzyme is not harmful, but when dosed with the prodrug ganciclovir, replicating TK-positive cells die, while non-dividing cells are spared. In preliminary studies, administration of ganciclovir to 3.6Col1-TK mice (which express TK in periosteal cells) results in a striking failure to make callus after fracture. In Aim 1, we will evaluate the temporal response to femur fracture in 3.6Col1-TK mice using radiography, microCT, histology, and mechanical testing. We hypothesize that 3.6Col1-TK mice treated with ganciclovir have greatly diminished callus formation and inferior biomechanical properties at 12 weeks compared to controls, indicative of an atrophic nonunion. In Aim 2, we propose to generate a novel floxed TK mouse line by CRISPR targeted knock-in of HSV-TK into the ROSA locus. By crossing these mice to any Cre mouse line, future studies will be able to ablate proliferating cells under temporal (ganciclovir) and lineage (Cre) control. Completion of these Aims will: a) provide direct evidence of the role of PPC proliferation in contributing to fracture callus; b) establish the 3.6Col1-TK mouse as new model of atrophic nonunion that can be used for future studies to test therapeutic interventions; and c) provide a new ROSA-TK mouse that will allow for future studies to determine the lineage of the cells that proliferate to form fracture callus.